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WO2021133724A1 - Cellules génétiquement corrigées à usage thérapeutique - Google Patents

Cellules génétiquement corrigées à usage thérapeutique Download PDF

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WO2021133724A1
WO2021133724A1 PCT/US2020/066388 US2020066388W WO2021133724A1 WO 2021133724 A1 WO2021133724 A1 WO 2021133724A1 US 2020066388 W US2020066388 W US 2020066388W WO 2021133724 A1 WO2021133724 A1 WO 2021133724A1
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cells
genodermatosis
population
ikcs
cell
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Anthony E. Oro
Marius Wernig
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • C12N5/063—Kereatinocyte stem cells; Keratinocyte progenitors
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    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
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    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • Pluripotential stem cells can self-renew and differentiate into somatic tissues, providing a source of cells for regenerative therapies.
  • Induced pluripotent stem cells iPSC
  • iPSC in particular can be generated from somatic tissues, thereby providing a supply of patient specific cells.
  • genetic manipulation of pluripotent cells and the subsequent transplantation allow genetic correction and selection, these techniques hold great promise in producing gene-modified cells and in treating genetic diseases.
  • the CRISPR-Cas9 system has enabled rapid genome editing in different species at a very high efficiency and specificity.
  • CRISPR-Cas9-mediated genome editing requires only a short single-guide RNA (sgRNA) to guide site-specific DNA recognition and cleavage, resulting in gene modification at a target locus via nonhomologous end joining (NHEJ)-mediated insertions/deletions (indels) or homology-directed repair (HDR) based on an exogenously supplied oligonucleotide.
  • sgRNA single-guide RNA
  • NHEJ nonhomologous end joining
  • indels homology-directed repair
  • HDR homology-directed repair
  • EB Epidermolysis bullosa
  • RDEB Recessive dystrophic epidermolysis bullosa
  • the COL7A1 locus encodes type VII collagen, the main component of the anchoring fibrils which tether the epidermis to the dermal tissue underneath.
  • Patients with RDEB develop large, severely painful blisters and open wounds, leaving affected children with painful chronic wounds over their body, and risk of death from cancer later in life.
  • Treatments are only supportive in nature and include prevention of trauma to the skin, promoting good wound care, treatment of skin infections, promotion and support of nutrition, treatment of anemia and skin cancer surveillance.
  • the basic underlying tenets of care for all EB patients are avoidance of blistering (by meticulous protective padding/dressings used on the skin), prevention of secondary infection, and promotion of an occlusive wound healing environment through the daily application of various types of non-adhesive dressings to large areas of the body.
  • Wound supplies can cost over ten thousand dollars per month.
  • substantial areas of denuded skin foster bacterial growth and can lead to infection.
  • chronic wounds and erosions contribute to anemia, pain, itch, and failure to thrive.
  • the cells are autologous with respect to an individual selected for treatment. In other embodiments the cells are allogeneic with respect to an individual selected for treatment.
  • the cells thus obtained may meet the requirements for clinical use, i.e. the cells are cGMP compatible.
  • Engineering iPSC allows patient samples to be obtained from cells such as fibroblasts, which may be more available than cells from affected tissues. Further, the iPSC samples can be stored frozen for long periods of time, and expanded in suitable numbers for multiple administrations of a tissue graft.
  • a composition of genetically corrected integration-free, cGMP compatible iPSC is provided.
  • the cells may be genetically corrected, for example, at a wide range of loci involved in the cause of disease, e.g. in muscle diseases, hematopoietic diseases, metabolic diseases, skin diseases, etc.
  • loci include, without limitation, dystrophin mutations involved in muscular dystrophy, for example at exon 51; hemoglobins involved in thalassemia or sickle cell anemia, for example beta-globin; targeting CCR5 for the treatment of HIV infection; Swedish APP5 in early onset Alzheimers disease; huntingtin mutations associated with Huntington’s disease; mutations associated with retinitis pigmentosa; LCA10 mutations associated with Leber congenital amaurosis; etc.
  • the manufacture of the genetically corrected iPSCs is optimized through the development of an integration-free, feeder-free, xeno-free, single clonal step method using preformed ribonuclear protein (RNPs) complexes with high-fidelity CAS9 and guide RNAs and mRNAs encoding reprogramming factors.
  • RNPs ribonuclear protein
  • the combination of footprint-free reprogramming and gene editing of patient somatic cells into a one-step process is dependent on the ability to efficiently generate iPSCs using non-integration-based methods and the development of high-efficiency gene editing.
  • the reprogramming to pluripotency utilizes synthetic capped mRNAs containing modified nucleobases (modified mRNA).
  • the reprogramming factors comprise a modified version of Oct4 fused with the MyoD transactivation domain (called M3O), Sox2, Klf4, cMyc, and Lin28A.
  • M3O MyoD transactivation domain
  • Sox2 Sox2
  • Klf4 cMyc
  • Lin28A Lin28A
  • the reprogramming is performed with a feeder-free system in xeno-free media.
  • the mRNA reprogramming method provides for robust iPS cell colony formation in patient fibroblast lines.
  • the reprogramming step above is combined with genetic correction in a single step. Genetic correction is performed with CRISPR-associated CAS9 nucleases, to induce a precise DNA double-strand break (DSB) at endogenous pathogenic genomic loci with high efficiency.
  • DSB DNA double-strand break
  • the advantages of this approach include the lack of integrating viruses or clonal selection, and the ability to perform both reprogramming and mutational correction in one step.
  • the CAS9 protein is delivered by mRNA transfection (mRNA) with RNP complexes, and may utilize high-fidelity CAS9 protein in RNP complexes.
  • the fibroblasts are transfected with RNPs and DNA oligonucleotides, e.g. from about 5 to about 25 pmole of guide mRNA and CAS9 protein. After from about 2 to about 6, about 3 to about 5, about 4 days in culture, cells are reprogrammed by repeated transfections with the mRNAs encoding the reprogramming factors in defined reprogramming media.
  • iPS cell colonies are screened for genetic correction, e.g. by droplet digital PCR, direct sequencings, etc.
  • Cells identified as having the correct genotype are expanded as iPSC and may be frozen for future use, or differentiated into somatic cells.
  • the cells are obtained from an individual with recessive dystrophic epidermolysis bullosa (RDEB), and a defect at the COL7A1 locus is corrected by an integration-free method to provide a normally functional collagen VII protein.
  • compositions and methods are provided for the treatment of EB in a human subject.
  • iPSCs Genetically corrected iPSCs obtained by the methods described herein can be grown in large quantities and be induced to differentiate into genetically corrected iPSC-derived keratinocyte stem cells, herein called iKCs.
  • iKCs resemble and functional like somatic patient keratinocytes, but are corrected at the COL7A1 locus.
  • a further improvement is provided by the selection of graftable iKCs for expansion.
  • Expression of CD49f (ITGA6) HI cells is shown to be an in-process potency marker that correlates with iKCs and tissue graftability.
  • ITGA6 is one of the earliest surface markers to arise in definitive mature keratinocytes and the ITGA6 bright population correlates with a K14 + , K18- expression basal keratinocyte phenotype. Importantly ITGA6 bright cells displayed enhanced graftability, resulting in an organotypic epidermis that displays proper polarity and differentiation. Selection may utilize one or more of flow cytometry, magnetic bead selection, and the like, with an affinity reagent, e.g. an antibody, specific for human CD49f. [0015] Clinical scaling and manufacturing of patient iKC sheets is achieved by differentiating patient-derived, COL7A1-corrected iPSCs.
  • the level of iKC COL7A1 expression is greater than normal human keratinocyte levels of expression. In some embodiments, the level of yKC COL7A1 expression is smaller, similar, or same as normal human keratinocyte levels of COL7A1 expression.
  • Included in the invention is an isolated population of iKCs engineered and selected by the methods described herein to express wild-type collagen VII, which may be provided in a pharmaceutical unit dose composition.
  • the subject is a human suffering from a genetic defect in collagen VII causing EB.
  • the genetic defect is RDEB.
  • a graftable epithelial sheet composition is provided.
  • a defined cell product can be obtained following in vitro expansion of iKC.
  • the process includes selection for desired cells types, for example by selecting cells with flow cytometry, magnetic bead selection, etc.
  • the composition desirably comprises greater than about 50% iKCs, which can be determined by, for example expression of CD104; and may be greater than 55%, greater than 60%, or more.
  • the composition desirably comprises less than about 35% fibroblast feeder cells, which can be determined by, for example expression of CD90; and may be less than 30%, less than 25%, or less.
  • the composition desirably comprises less than about 1% undifferentiated iPSCs, which can be determined by, for example expression of Tra1-60; and may be less than 0.5%%, less than 0.1%, or less.
  • the defined cell composition is placed in culture, grown to confluence and then released and delivered to the operating room.
  • a method for treatment of EB comprising, consisting essentially of, or yet further consisting of, producing a genetically corrected population of iPSC from a subject suffering from EB by the methods described herein, differentiating the iPSC to iKCs and reintroducing the iKC-derived sheets into the individual.
  • the iKCs are selected for expression of CD49f prior to manufacture of keratinocyte sheets for grafting.
  • the genetically corrected iKCs are cultured to generate a sheet of from about 25 cm 2 to about 100 cm 2 for grafting.
  • the iKCs sheets are placed on uninfected, eroded, and/or scarred wound sites that lacks clinical evidence of squamous cell carcinoma (SCC). Wound sites may be from about 50 cm 2 , from about 100 cm 2 , and/or from about 200 cm 2 .
  • wounds are generated for grafts. In some such embodiments, the wound is electrocauterized to ablate residual non-corrected wound bed keratinocytes.
  • the disclosure provides a pharmaceutical composition comprising, consisting essentially of, or yet further consisting of an iKC sheet, which comprises, consists essentially of, or yet further consists of iKCs genetically corrected with an integration- free method.
  • the iKC sheet displays proper polarity and differentiation for epidermal grafts.
  • the iKC sheet is placed on a bioengineered skin equivalent.
  • the iKC sheet is placed on an acellular matrix, a collagen matrix, an ECM protein or chemical layer, or a biocompatible mesh.
  • the acellular matrix is made of human and/or animal dermis.
  • the biocompatible mesh is made of thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicon, or any combination thereof.
  • FIG. 1 Characterization of corrected IPS Intermediates.
  • any embodiment of any of the present methods, devices, and systems may consist of, or consist essentially of—rather than comprise/include/contain/have—the described steps and/or features.
  • the term “consisting of” or “consisting essentially of” may be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb.
  • pluripotency and pluripotent stem cells it is meant that such cells have the ability to differentiate into all types of cells in an organism.
  • induced pluripotent stem cell encompasses pluripotent cells, that, like embryonic stem (ES) cells, can be cultured over a long period of time while maintaining the ability to differentiate into all types of cells in an organism, but that, unlike ES cells (which are derived from the inner cell mass of blastocysts), are derived from differentiated somatic cells, that is, cells that had a narrower, more defined potential and that in the absence of experimental manipulation could not give rise to all types of cells in the organism.
  • iPS cells By “having the potential to become iPS cells” it is meant that the differentiated somatic cells can be induced to become, i.e. can be reprogrammed to become, iPS cells.
  • the somatic cell can be induced to dedifferentiate so as to establish cells having the morphological characteristics, growth ability and pluripotency of pluripotent cells.
  • iPS cells have an hESC-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei.
  • iPS cells express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42.
  • the iPS cells are capable of forming teratomas. In addition, they are capable of forming or contributing to ectoderm, mesoderm, or endoderm tissues in a living organism.
  • Modified mRNA (mmRNA) composition In the methods of the invention, one form of nuclear reprogramming is accomplished by contacting somatic cells with a cocktail of mmRNA encoding reprogramming factors.
  • the modified mRNA may be provided as a purified IVT transcript.
  • reprogramming factors are known and used in the art, including, for example, for nuclear reprogramming of a somatic cell to pluripotency, one may use a cocktail comprising the five reprogramming factors (OKSML; Oct 4, KLF4, Sox2, cMyc and Lin28) in equimolar quantities except for Oct 4.
  • Preferred is the use of a fusion gene between O ct4 and the transactivation domain (TAD) of MyoD (M3O) (see Hirai et al.)
  • mmRNA may comprise one or more non-natural nucleotides.
  • the nucleoside modification may include a compound selected from the group consisting of pyridin- 4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hy-droxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1 - carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethy1-2-thiouridine, 1-tau- rinomethy1-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2 -thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pse
  • the modifications are independently selected from the group consisting of 5-methylcytosine, pseudouridine and 1-methylpseudouridine.
  • Two modifications of the nucleic acid molecule may be located on nucleosides of the modified nucleic acid molecule.
  • the modified nucleosides may be selected from 5-methylcytosine and pseudouridine.
  • a 5’ cap structure can be a native 7-methylguanylate cap, or a cap analog, for example anti-reverse cap analog (ARCA), 3 ⁇ -O-Me-m7G(5')ppp(5')G, (m7G(5 ⁇ )ppp(5 ⁇ )G), Cap0, Cap1, inosine, N1-methyl-guanosine, 2' fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2- amino-guanosine, LNA-guanosine, 2-azido-guanosine, etc.
  • a 5’ cap structure can be a native 7-methylguanylate cap, or a cap analog, for example anti-reverse cap analog (ARCA), 3 ⁇ -O-Me-m7G(5')ppp(5')G, (m7G(5 ⁇ )ppp(5 ⁇ )G), Cap0, Cap1, inosine, N1-methyl-
  • Reprogramming factors refers to one or a cocktail of biologically active polypeptides that act on a cell to alter transcription, and which upon expression, reprogram a somatic cell a different cell type, or to multipotency or to pluripotency.
  • the reprogramming factors are provided as mmRNA encoding the polypeptides described below.
  • the reprogramming factor is a transcription factor, including without limitation, M3O; Sox2; Klf4; c-Myc; and Nanog.
  • Lin28 is an mRNA-binding protein thought to influence the translation or stability of specific mRNAs during differentiation.
  • a mmRNA composition may encode one or more biologically active reprogramming factors.
  • the composition may comprise at least about 10 ng/ ⁇ l each mmRNA specificity, (i.e. encoding each reprogramming factor), at least about 50 ng/ ⁇ l; at least about 100 ng/ ⁇ l, at least about 200 ng/ ⁇ l, at least about 250 ng/ ⁇ l, at least about 300 ng/ ⁇ l, or more.
  • a Klf4 polypeptide is a polypeptide comprising the amino acid sequence that is at least 70% identical to the amino acid sequence of human Klf4, i.e., Kruppel-Like Factor 4 the sequence of which may be found at GenBank Accession Nos.
  • Klf4 polypeptides e.g. those that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or 100% identical to the sequence provided in GenBank Accession No. NM_004235.
  • the nucleic acids that encode them find use as a reprogramming factor in the present invention.
  • a c-Myc polypeptide is a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of human c-Myc, i.e., myelocytomatosis viral oncogene homolog, the sequence of which may be found at GenBank Accession Nos.
  • a Nanog polypeptide is a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of human Nanog, i.e., Nanog homeobox, the sequence of which may be found at GenBank Accession Nos. NP_079141 and NM_024865.
  • Nanog polypeptides e.g. those that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or 100% identical to the sequence provided in GenBank Accession No. NM_024865.
  • the nucleic acids that encode them find use as a reprogramming factor in the present invention.
  • a Lin-28 polypeptide is a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of human Lin-28, i.e., Lin-28 homolog of C. elegans, the sequence of which may be found at GenBank Accession Nos. NP_078950 and NM_024674. Lin-28 polypeptides, e.g.
  • Oct3/4 polypeptide is a polypeptide comprising an amino acid sequence that is at least 70% identical to the amino acid sequence of human Oct 3/4, also known as Homo sapiens POU class 5 homeobox 1 (POU5F1) the sequence of which may be found at GenBank Accession Nos. NP_002692 and NM_002701. Oct3/4 polypeptides, e.g.
  • a Sox2 polypeptide is a polypeptide comprising the amino acid sequence at least 70% identical to the amino acid sequence of human Sox2, i.e., sex-determining region Y-box 2 protein, the sequence of which may be found at GenBank Accession Nos. NP_003097 and NM_003106. Sox2 polypeptides, e.g.
  • primary cells those that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 95%, 97%, 99%, or 100% identical to the sequence provided in GenBank Accession No. NM_003106.
  • the nucleic acids that encode them find use as a reprogramming factor in the present invention.
  • the terms “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cell cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture.
  • starting cell population refers to a somatic cell, usually a primary, or non-transformed, somatic cell, such as a fibroblast, which undergoes nuclear reprogramming and genetic correction by the methods of the invention.
  • the starting cell population may be of any mammalian species, but particularly including human cells. Sources of starting cell populations include individuals desirous of cellular therapy, individuals having a genetic defect of interest for study, and the like.
  • human cells obtained from a subject for the purpose of nuclear reprogramming and genetic correction may be chosen from any human cell type, including fibroblast cells, adipose tissue cells, mesenchymal cells, bone marrow cells, stomach cells, liver cells, epithelial cells, nasal epithelial cells, mucosal epithelial cells, follicular cells, connective tissue cells, muscle cells, bone cells, cartilage cells, gastrointestinal cells, splenic cells, kidney cells, lung cells, testicular cells, nervous tissue cells, etc.
  • the human cell type is a fibroblast, which may be conveniently obtained from a subject by a punch biopsy.
  • the cells are obtained from subjects known or suspected to have a copy number variation (CNV) or mutation of the gene of interest.
  • the cells are from a patient presenting with idiopathic/sporadic form of the disease.
  • the cells are from a non-human subject.
  • the cells are then reprogrammed and genetically corrected, and may be subsequently differentiated to adopt a specific cell fate, such as endodermal cells, neuronal cells, for example dopaminergic, cholinergic, serotonergic, GABAergic, or glutamatergic neuronal cell; pancreatic cells, e.g. islet cells, muscle cells including without limitation cardiomyocytes, hematopoietic cells, and the like.
  • CRISPR Class 2 Clustered Regularly Interspaced Short Palindromic Repeat
  • Engineered CRISPR systems contain two components: a guide RNA (gRNA or sgRNA) and a CRISPR-associated endonuclease (Cas protein).
  • gRNA guide RNA
  • Cas protein CRISPR-associated endonuclease
  • the gRNA is a short synthetic RNA composed of a scaffold sequence necessary for Cas-binding and a user-defined ⁇ 20 nucleotide spacer that defines the genomic target to be modified.
  • gRNA guide RNA
  • Cas protein CRISPR-associated endonuclease
  • the Cas9 protein and the gRNA form a ribonucleoprotein complex through interactions between the gRNA scaffold and surface-exposed positively-charged grooves on Cas9.
  • Cas9 undergoes a conformational change upon gRNA binding that shifts the molecule from an inactive, non-DNA binding conformation into an active DNA-binding conformation.
  • the spacer region of the gRNA remains free to interact with target DNA.
  • Any genomic sequence of about 20 nucleotides can be targeted for protection, provided the sequence is unique compared to the rest of the genome and target is present immediately adjacent to a Protospacer Adjacent Motif (PAM).
  • PAM Protospacer Adjacent Motif
  • the PAM sequence serves as a binding signal for Cas9, but the exact sequence depends on which Cas protein is used. Cas9 will only cleave a given locus if the gRNA spacer sequence shares sufficient homology with the target DNA. Once the Cas9-gRNA complex binds a putative DNA target, the seed sequence (8-10 bases at the 3′ end of the gRNA targeting sequence) will begin to anneal to the target DNA. If the seed and target DNA sequences match, the gRNA will continue to anneal to the target DNA in a 3′ to 5′ direction.
  • Cas9 undergoes a second conformational change upon target binding that positions the nuclease domains, called RuvC and HNH, to cleave opposite strands of the target DNA.
  • the end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA ( ⁇ 3- 4 nucleotides upstream of the PAM sequence).
  • CRISPR specificity can also be increased through modifications to Cas9.
  • High fidelity Cas9's include eSpCas9(1.1) and SpCas9-HF1.
  • eSpCas9(1.1) contains alanine substitutions that weaken the interactions between the HNH/RuvC groove and the non-target DNA strand, preventing strand separation and cutting at off-target sites.
  • SpCas9-HF1 lowers off-target editing through alanine substitutions that disrupt Cas9's interactions with the DNA phosphate backbone.
  • HypaCas9 contains mutations in the REC3 domain that increase Cas9 proofreading and target discrimination. All three high fidelity enzymes generate less off-target editing than wild type Cas9.
  • a DNA repair template containing the desired sequence is delivered into the cell type of interest with the gRNA(s) and Cas9.
  • the repair template contains the desired edit as well as additional homologous sequence immediately upstream and downstream of the target (termed left & right homology arms.) The length of each homology arm is dependent on the size of the change being introduced, with larger insertions requiring longer homology arms.
  • the repair template may be a single-stranded oligonucleotide, double-stranded oligonucleotide, or a double- stranded DNA plasmid.
  • the term “efficiency of reprogramming and genetic correction” may be used to refer to the ability of a cells to give rise to iPS cell colonies when contacted with CRISPR/cas9 for genetic correction in a single step method with reprogramming factors. The efficiency of reprogramming with the methods of the invention vary with the particular combination of somatic cells, method of introducing reprogramming factors, and method of culture following induction of reprogramming.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
  • the terms "individual,” “subject,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • Keratinocytes Keratinocytes.
  • Keratinocyte cells are found in the deepest basal layer of the stratified epithelium that comprises the epidermis, and are sometimes referred to as basal cells or basal keratinocytes. It is known that 95% of the cells in the epidermis are keratinocytes. Squamous keratinocytes are also found in the mucosa of the mouth and esophagus, as well as the corneal, conjunctival and genital epithelia. [0022] Keratinocytes are maintained at various stages of differentiation in the epidermis and are responsible for forming tight junctions with the nerves of the skin. They also keep Langerhans cells of the epidermis and lymphocytes of the dermis in place.
  • Keratinocyte stem cells reside in the basal layer of the epidermis, which is the lowest layer of the stratified epithelia. These cells divide to give rise to transient amplifying cells which divide further, and differentiate, as they move upwards in the epidermis. The differentiating cells produce compounds and other proteins which are critical to the integrity of the outermost layer of the skin, the stratum corneum.
  • the keratinocytes in the stratum corneum are dead squamous cells that are no longer multiplying. Once keratinocytes reach the corneum, they are said to be keratinazed, or cornified, creating the tough outer layer of skin.
  • Induced Keratinocytes are keratinocyte stem cell-like cells that resemble those that divide to give rise to the different layers of the stratified epidermis. They derive from pluripotent cells (ES or iPS) through the addition of developmental morphogens such as RA and BMP, and like somatic keratinocyte stem cells, can provide a skin barrier when grafted into a wound.
  • the major proteins found in keratinocytes are keratins. These proteins form the cytoskeleton of keratinocytes, and keratin expression changes as transient amplifying cells differentiate and move to the most superficial stratum corneum.
  • Selection for keratinocyte stem cells are keratinocyte stem cell-like cells that resemble those that divide to give rise to the different layers of the stratified epidermis. They derive from pluripotent cells (ES or iPS) through the addition of developmental morphogens such as RA and BMP, and like somatic keratinocyte stem cells, can
  • the graftable keratinocytes may be selected for CD49f high expression.
  • the CD49f (ITGA6) protein product is the integrin alpha chain alpha 6.
  • the reference sequence for the human protein may be accessed at Genbank, NP_000201.
  • Coupling Efficiency (CE) refers to the ratio of the graftable iKCs (CD49f-bright) to corrected iPS at the start of the differentiation process. This ratio is useful to measure the efficiency of the manufacturing process.
  • Expansion Coefficient refers to the number of graftable iKCs present after serial passaging compared to the original number of iKCs in the Clinimacs purification.
  • Selection for cells may use conventional methods.
  • Cells of interest i.e. cells expressing the marker of choice, may be enriched for, that is, separated from the rest of the cell population, by a number of methods that are well known in the art.
  • flow cytometry e.g. fluorescence activated cell sorting (FACS)
  • FACS fluorescence activated cell sorting
  • selection of the cells may be effected by flow cytometry.
  • each cell is recorded as a data point having a particular intensity of staining.
  • These data points may be displayed according to a log scale, where the unit of measure is arbitrary staining intensity.
  • the brightest stained cells in a sample can be as much as 4 logs more intense than unstained cells.
  • the "low" positively stained cells have a level of staining above the brightness of an isotype matched control, but are not as intense as the most brightly staining cells normally found in the population.
  • An alternative control may utilize a substrate having a defined density of marker on its surface, for example a fabricated bead or cell line, which provides the positive control for intensity.
  • Other methods of separation i.e. methods by which selection of cells may be effected, based upon markers include, for example, magnetic activated cell sorting (MACS), immunopanning, and laser capture microdissection.
  • the affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above.
  • peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptors; effector and receptor molecules, and the like.
  • Antibodies and T cell receptors may be monoclonal or polyclonal, and may be produced by transgenic animals, immunized animals, immortalized human or animal B-cells, cells transfected with DNA vectors encoding the antibody or T cell receptor, etc. The details of the preparation of antibodies and their suitability for use as specific binding members are well-known to those skilled in the art. [0032] Of particular interest is the use of antibodies as affinity reagents. Conveniently, these antibodies are conjugated with a label for use in separation.
  • Labels include magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes, which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type.
  • Fluorochromes that find use include phycobiliproteins, e.g. phycoerythrin and allophycocyanins, fluorescein and Texas red. Frequently each antibody is labeled with a different fluorochrome, to permit independent sorting for each marker.
  • the antibodies are added to a suspension of cells, and incubated for a period of time sufficient to bind the available cell surface antigens.
  • the incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient concentration of antibodies in the reaction mixture, such that the efficiency of the separation is not limited by lack of antibody. The appropriate concentration is determined by titration.
  • the medium in which the cells are separated will be any medium that maintains the viability of the cells. A preferred medium is phosphate buffered saline containing from 0.1 to 0.5% BSA.
  • Genodermatoses are genetic diseases with cutaneous expression. They are various (around 400) and almost all rare. Their prevalence is between 1:6000 and 1:500000. They usually break out at birth or early in life and severely affect children.
  • Ectodermal Dysplasia examples include, for example, Ectodermal Dysplasia; Hypohidrotic ectodermal dysplasia (ED1 is the causative gene); Hidrotic ectodermal dysplasia (GJB6 is the causative gene); White sponge nevus (keratin-4 or keratin-13 is the causative gene); Hereditary, benign, intraepithelial-dyskeratosis (a segment of DNA localized at 4q35 is duplicated resulting in triple alleles for 2 linked markers); Pachyonychia congenita (specific mutations in the keratin 16 gene-Jadassohn–Lewandowsky type, mutations of the keratin 17 gene are associated with the Jackso–Lawler form); Dyskeratosis congenita
  • a condition for treatment is Netherton syndrome, which is a disorder that affects the skin, hair, and immune system. Newborns with Netherton syndrome have skin that is red and scaly (ichthyosiform erythroderma), and the skin may leak fluid. Some affected infants are born with a tight, clear sheath covering their skin called a collodion membrane. This membrane is usually shed during the first few weeks of life. Because newborns with this disorder are missing the protection provided by normal skin, they are at risk of becoming dehydrated and developing infections in the skin or throughout the body (sepsis), which can be life-threatening. Affected babies may also fail to grow and gain weight at the expected rate (failure to thrive).
  • Netherton syndrome is caused by mutations in the SPINK5 gene, which encodes LEKT1 protein.
  • LEKT1 is a serine peptidase inhibitor found in the skin and in the thymus. LEKT1 controls the activity of serine peptidases in the epidermis, particularly the stratum corneum. Serine peptidase enzymes are involved in normal skin shedding by helping to break the connections between cells of the stratum corneum. LEKT1 is also involved in normal hair growth, the development of lymphocytes in the thymus, and the control of peptidases that trigger immune system function.
  • Epidermolysis Bullosa Conditions of interest for treatment with engineered keratinocytes include, without limitation, various forms of Epidermolysis Bullosa, including acquired and congenital forms, the latter of which may be recessive or dominant.
  • Currently classified into four main subtypes EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, mainly based on the level of skin cleavage), the spectrum of EB extends to more than 30 clinical subtypes with pathogenic mutations in at least 18 distinct genes.
  • DST-e coding for epidermal dystonin, also known as the 230 kDa bullous pemphigoid antigen, BP230
  • EXPH5 coding for exophilin-5, also known as Slac2-b
  • ITGA3 coding for the integrin alpha-3 subunit
  • Dystrophic Epidermolysis Bullosa includes three subtypes: recessive DEB, severe generalized (RDEB-sev gen) (formerly called Hallopeau-Siemens type (RDEB-HS); recessive DEB, generalized other (RDEB-O) (formerly called non-Hallopeau-Siemens type (RDEB-non- HS); and dominant DEB (DDEB).
  • RDEB-sev gen blisters affecting the whole body may be present in the neonatal period. Oral involvement may lead to mouth blistering, fusion of the tongue to the floor of the mouth, and progressive diminution of the size of the oral cavity.
  • Esophageal erosions can lead to webs and strictures that can cause severe dysphagia. Consequently, severe nutritional deficiency and secondary problems are common. Corneal erosions can lead to scarring and loss of vision. Blistering of the hands and feet followed by scarring fuses the digits into “mitten” hands and feet, a hallmark of this disorder. The lifetime risk of aggressive squamous cell carcinoma is over 90%. In DDEB, blistering is often mild and limited to hands, feet, knees, and elbows, but nonetheless heals with scarring. Dystrophic nails, especially toenails, are common and may be the only manifestation of DDEB. [0041] Conventional treatment of manifestations is primarily supportive, including wound dressing and nutritional support. Occupational therapy may help prevent hand contractures.
  • Keratinocytes engineered to express wild-type C7 can find use in therapy for Dystrophic Epidermolysis Bullosa.
  • EBA Epidermolysis Bullosa
  • Circulating autoantibodies in patients with EBA recognize epitopes in type VII collagen molecules, and molecular cloning of the type VII collagen cDNAs have provided the tools to identify the predominant immunoepitopes within the amino-terminal NC-1 domain of type VII collagen.
  • NC-1(VII) domain The antigenic properties of the NC-1(VII) domain are further highlighted by the fact that monoclonal antibodies, such as H3A and L3D, which are in clinical use to map type VII collagen in the skin of patients with inherited forms of EB, also identify epitopes in this portion of the protein. In addition to circulating autoantibodies recognizing type VII collagen epitopes in EBA, bullous lesions in some patients with systemic lupus erythematosus have also been associated with anti- type VII collagen antibodies. [0044] Collagen.
  • collagen refers to compositions in which at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more of the protein present is collagen in a triple helical configuration.
  • the folding of the individual ⁇ chains into the triple-helical conformation is predicated upon the characteristic primary sequence, including repeating Gly-X-Y triplet sequences.
  • Collagens are widely found in vertebrate species and have been sequenced for many different species. Due to the high degree of sequence similarity between species, collagen from different species can be used for biomedical purposes, e.g., between mammalian species, although the human protein may be preferred.
  • FACIT collagens include types IX, XII, XIV, XIX, XX, and XXI.
  • types IX, XII, XIV, XIX, XX, and XXI include types IX, XII, XIV, XIX, XX, and XXI.
  • Collagen VII (COL7A1, Chromosome 3, NC_000003.10 (48576510..48607689, complement)) is of particular interest.
  • Type VII collagen is a major component of anchoring fibrils.
  • Type VII collagen is a long, 424 nm, triple-helical domain with flanking non-collagenous sequences.
  • Type VII collagen molecules include a central collagenous, triple-helical segment flanked by the non-collagenous NC-1 and NC-2 domains. Unlike interstitial collagens, the repeating Gly-X-Y sequence is interrupted by 19 imperfections due to insertions or deletions of amino acids in the Gly-X-Y repeat sequence. Most notably, in the middle of the triple-helical domain, there is a 39-amino acid non-collagenous “hinge” region which is susceptible to proteolytic digestion with pepsin.
  • the amino-terminal NC-1 domain of type VII includes sub-modules with homology to known adhesive proteins, including segments with homology to cartilage matrix protein (CMP), nine consecutive fibronectin type III- like (FN-III) domains, a segment with homology to the A domain of von Willebrand factor, and a short cysteine and proline-rich region.
  • CMP cartilage matrix protein
  • FN-III fibronectin type III- like domains
  • the carboxy-terminal non- collagenous domain, NC-2 is relatively small, ⁇ 30kDa, and it contains a segment with homology to Kunitz protease inhibitor molecule.
  • the human type VII collagen gene, COL7A1 has a complex structure with a total of 118 separate exons.
  • the gene is, however, relatively compact, and most of the introns are relatively small; consequently, the size of the entire human COL7A1 gene is only ⁇ 32 kb, encoding a messenger RNA of ⁇ 8.9 kb.
  • COL7A1 has been mapped to the short-arm of human chromosome 3, region 3p21.1.
  • the type VII collagen gene structure and the encoded primary sequence of the protein are well conserved, and for example, the mouse gene shows 84.7% homology at the nucleotide and 90.4% identity at the protein level.
  • Type VII collagen is synthesized both by epidermal keratinocytes and dermal fibroblasts in culture.
  • pro- ⁇ 1(VII) polypeptides Upon synthesis of complete pro- ⁇ 1(VII) polypeptides, three polypeptides associate through their carboxy-terminal ends to a trimer molecule which in its collagenous portion folds into the triple-helical formation. The triple-helical molecules are then secreted to the extracellular milieu where two types of VII collagen molecules align into an anti-parallel dimer with the amino- terminal domains present at both ends of the molecule. This dimer assembly is accompanied by proteolytic removal of a portion of the carboxy-terminal end of both type VII collagen molecules and stabilization by inter-molecular disulfide bond formation. Subsequently, a large number of these anti-parallel dimers aggregate laterally to form anchoring fibrils.
  • Glycine substitution mutations in the triple helical domain of COL7A1 predominate in Dominant Dystrophic Epidermolysis Bullosa (DDEB). Mutations p.Gly2034Arg and p.Gly2043Arg are the most common DDEB-causing mutations, making up 50% of the dominant mutations reported in the largest US cohort. Glycine substitutions as well as other amino acid substitutions and splice junction mutations outside of this region may also be found in dominant DEB. [0050] More than 400 recessive DEB-causing mutations spanning the entire gene have been described for all forms of DEB.
  • a “native sequence” polypeptide is one that has the same amino acid sequence as a polypeptide derived from nature. Such native sequence polypeptides can be produced by recombinant means according to the methods set forth herein. Thus, a native sequence polypeptide can have the amino acid sequence of, e.g., naturally occurring human polypeptide, murine polypeptide, or polypeptide from any other mammalian species, and the like.
  • native sequence collagen VII protein includes the native proteins with or without the initiating N- terminal methionine (Met).
  • a “variant” polypeptide means a biologically active polypeptide as defined below having less than 100% sequence identity with a native sequence polypeptide. Such variants include polypeptides wherein one or more amino acid residues are added at the N- or C-terminus of, or within, the native sequence; from about one to forty amino acid residues are deleted, and optionally substituted by one or more amino acid residues; and derivatives of the above polypeptides, wherein an amino acid residue has been covalently modified so that the resulting product has a non-naturally occurring amino acid.
  • a biologically active collagen VII variant will have an amino acid sequence having at least about 90% amino acid sequence identity with a native sequence collagen VII polypeptide, preferably at least about 95%, more preferably at least about 99%.
  • a “functional derivative” of a native sequence collagen, VII polypeptide is a compound having a qualitative biological property in common with a native sequence collagen VII polypeptide. “Functional derivatives” include, but are not limited to, fragments of a native sequence and derivatives of a native sequence collagen VII polypeptide and its fragments, provided that they have a biological activity in common with a corresponding native sequence collagen VII polypeptide. The term “derivative” encompasses both amino acid sequence variants of collagen VII polypeptide and covalent modifications thereof.
  • wound bed refers to the uppermost viable layer of wound.
  • the wound bed is covered by slough or eschar.
  • the wound bed can be assessed for presence of granulation tissue fibrin slough, eschar, bone, tendon, and/or other underlying structures.
  • genetic modification refers to a process of altering a gene of an organism or inserting a gene from one organism into another organism.
  • the genetic modification comprises, consists essentially of, or yet consists of insertion, deletion, and/or mutation.
  • insertion means addition of one or more nucleotide base pairs into a nucleotide sequence.
  • deletion refers to a part of a chromosome or a nucleotide sequence that is removed or missing.
  • mutation is alteration of nucleotide sequence (e.g., DNA sequence). The mutation can occur in various sizes, including, but not limited to, a single base pair (i.e., point mutation), several base pairs, or up to a large segment of chromosome.
  • conserve genetic modification refers to genetic modification that maintain same or similar biochemical properties of a polypeptide encoded by the genetically modified gene. For example, both aspartic acid and glutamic acid are both small, negatively charged residues.
  • a starting population of somatic cells e.g. fibroblasts
  • somatic cells are genetically corrected at a locus of interest, and reprogrammed to pluripotency in a single step for GMP compatible integration- free, xeno-free, feeder-free iPSC, which iPSC can provide a source of further differentiated cells for treatment of genetic disorders.
  • Engineering iPSC allows patient samples to be obtained from cells such as fibroblasts, which may be more available than cells from affected tissues.
  • the iPSC samples can be stored frozen for long periods of time, and expanded in suitable numbers for multiple administrations of a tissue graft.
  • the starting population of somatic cells conveniently fibroblasts although other cell types also find use, are genetically corrected by CRISPR technology as described above.
  • the cells are contacted with a population of mmRNA encoding reprogramming factors, as defined above, in a combination and quantity sufficient to reprogram the cell to pluripotency.
  • Reprogramming factors may be provided to the somatic cells individually or as a single composition, that is, as a premixed composition, of reprogramming factors.
  • telomeres are typically required, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 transfections, which can be repeated daily, seemi- daily, every two days, etc.
  • a set of at least three mRNA encoding reprogramming factors is added, e.g., Oct3/4 or a modified variant thereof, Sox2, and Klf4, c-myc, nanog or lin28.
  • a set of five mRNA encoding reprogramming factors is provided to the cells e.g., Oct4 fused with the MyoD transactivation domain (called M3O), Sox2, Klf4, cMyc, and Lin28A.
  • Methods for introducing the mmRNA encoding reprogramming factors to somatic cells include transfection, lipofection, electroporation, exosomal delivery and the like, as known in the art. Following introduction of the mmRNA, cells are incubated for about 30 minutes to about 72 hours, e.g., 2 hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours 36 hours, 48 hours, 60 hours, 72 hours, or any other period from about 30 minutes to about 72 hours.
  • the reprogramming factors may be provided to the subject cells for about one to about 4 weeks, e.g. from about two to about 3 weeks.
  • the dose of mmRNA encoding reprogramming factors will vary with the nature of the cells, the factors, the culture conditions, etc. In some embodiments the dose will be from about 1 nM to about 1 ⁇ M for each factor, more usually from about 10 nM to about 500 nM, or around about 100 to 200 nM. In embodiments where an additional activator is used, the cells are initially exposed during exposure to the reprogramming actors for at least about 1 day, at least about 2 days, at least about 4 days, at least about 6 days or one week, and may be exposed for the entire reprogramming process, or less.
  • the dose will depend on the specific agonist, but may be from about 1 ng/ml to about 1 ⁇ g/ml, from about 10 ng/ml to about 500 ng/ml.
  • the somatic cells may be maintained in a culture medium in the absence of feeder layers, i.e. lacking somatic cells other than those being induced to pluripotency.
  • Feeder layer free cultures may utilize a protein coated surface, e.g. matrigel, etc.
  • the medium may also be free of xenogeneic (non-human) protein factors such as fetal bovine serum.
  • iPS cells induced to become such by the methods of the invention have an hESC-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei.
  • the iPS cells may express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3/4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, FoxD3, GDF3, Cyp26a1, TERT, and zfp42.
  • the iPS cells are capable of forming teratomas.
  • the iPS cells produced by the above methods may be used for reconstituting or supplementing differentiating or differentiated cells in a recipient.
  • the induced cells may be differentiated into cell-types of various lineages. Examples of differentiated cells include any differentiated cells from ectodermal (e.g., neurons and fibroblasts), mesodermal (e.g., cardiomyocytes), or endodermal (e.g., pancreatic cells) lineages.
  • the differentiated cells may be one or more: pancreatic beta cells, neural stem cells, neurons (e.g., dopaminergic neurons), oligodendrocytes, oligodendrocyte progenitor cells, hepatocytes, hepatic stem cells, astrocytes, myocytes, hematopoietic cells, or cardiomyocytes.
  • neurons e.g., dopaminergic neurons
  • oligodendrocytes oligodendrocyte progenitor cells
  • hepatocytes e.g., hepatic stem cells
  • astrocytes e.g., myocytes, hematopoietic cells, or cardiomyocytes.
  • HSCs hematopoietic stem cells
  • the induced cells, or cells differentiated from the induced cells may be used as a therapy to treat disease (e.g., a genetic defect).
  • the therapy may be directed at treating the cause of the disease; or alternatively, the therapy may be to treat the effects of the disease or condition.
  • the induced cells may be transferred to, or close to, an injured site in a subject; or the cells can be introduced to the subject in a manner allowing the cells to migrate, or home, to the injured site.
  • the transferred cells may advantageously replace the damaged or injured cells and allow improvement in the overall condition of the subject. In some instances, the transferred cells may stimulate tissue regeneration or repair.
  • the transferred cells may be cells differentiated from induced cells.
  • the transferred cells also may be multipotent stem cells differentiated from the induced cells.
  • the transferred cells may be induced cells that have not been differentiated.
  • the number of administrations of treatment to a subject may vary. Introducing the induced and/or differentiated cells into the subject may be a one-time event; but in certain situations, such treatment may elicit improvement for a limited period of time and require an on-going series of repeated treatments. In other situations, multiple administrations of the cells may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.
  • the cells may be introduced to the subject via any of the following routes: parenteral, intravenous, intraarterial, intramuscular, subcutaneous, transdermal, intratracheal, intraperitoneal, or into spinal fluid.
  • the differentiated cells may be administered in any physiologically acceptable medium or device. They may be provided alone or with a suitable substrate or matrix, e.g. to support their growth and/or organization in the tissue to which they are being transplanted. Usually, at least 1x10 5 cells will be administered, preferably 1x10 6 or more.
  • the cells may be introduced by injection, catheter, sprayer (j.e. Spray-on-skin) or the like.
  • the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use on thawing. If frozen, the cells will usually be stored in a 10% DMSO, 50% FCS, 40% RPMI 1640 medium. Once thawed, the cells may be expanded by use of growth factors and/or stromal cells associated with progenitor cell proliferation and differentiation.
  • the genetically corrected iPSC are differentiated to keratinocytes, optionally being selected for CD49f expression prior to manufacture of the final epithelial sheet, and used for treatment of EB. In such methods, a keratinocyte sheet is used as a graft on an EB wound.
  • the wound is free of non-corrected wound bed keratinocytes.
  • the wound is treated to ablate non-corrected wound bed keratinocytes.
  • the subject suffers from Recessive Dystrophic Epidermolysis Bullosa (RDEB).
  • RDEB Recessive Dystrophic Epidermolysis Bullosa
  • the subject is human.
  • the keratinocyte sheet is placed on an acellular matrix, a collagen matrix, or a biocompatible mesh.
  • the biocompatible mesh is made of thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicon, or any combination thereof.
  • the skin cells comprise, alternatively consist essentially of, or yet further consist of keratinocytes.
  • the biocompatible mesh can be made from non-resorbable materials, including, but not limited to, biocompatible metals such as titanium alloys, stainless steel, cobalt- chromium alloys, and nickel-titanium alloys.
  • the layer of biocompatible mesh can be made from non-resorbable polymeric materials, including, but not limited to, thermoplastic resins, polyethylenes, ultra-high molecular weight polyethylene, high molecular weight polyolefins, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, any polymer or aliphatic hydrocarbons containing one or more double bonds, any other appropriate porous materials, or any other appropriate porous material that can be bent or otherwise formed into a shape.
  • non-resorbable polymeric materials including, but not limited to, thermoplastic resins, polyethylenes, ultra-high molecular weight polyethylene, high molecular weight polyolefins, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, any polymer or ali
  • the biocompatible mesh can be composed of a synthetic or biological resorbable polymeric material, including, but not limited to, polyglycolic acid, poly-L-lactic acid (PLLA), poly-D,L-lactic acid (PDLA), trimethylene carbonate (TMC), poly-£-caprolactone, poly-P- dioxanone, copolymers of lactide and glycolide (PLGA), polyhydroxy-3-butyrate, collagen, hyaluronic acid, silk, biocellulose, other protein-based polymers, polysaccharides, poly(DTE carbonate), polyarylates, blends of PLLA, PLDA, or PLGA with TMC and other combinations of these polymers.
  • PLLA poly-L-lactic acid
  • PDLA poly-D,L-lactic acid
  • TMC trimethylene carbonate
  • PLGA poly-£-caprolactone
  • PLGA polyhydroxy-3-butyrate
  • collagen hyaluronic acid
  • silk biocellulose
  • biocellulose other protein-based
  • the biocompatible mesh is made of thermoplastic resin, polyethylene, ultra-high molecular weight polyethylene, high molecular weight polyolefin, uncoated monofilament polypropylene, polyether ether ketone, polyethylene terephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, nylon, silicon, or any combination thereof.
  • composition comprising, alternatively consisting essentially of, or yet further consisting of a keratinocyte sheet, wherein the keratinocyte sheet is prepared by a process comprising, alternatively consisting essentially of, or yet further consisting of the steps of: obtaining a population of fibroblasts from a subject; genetically correcting and reprogramming the cells in a single step method; differentiating the reprogrammed cells to form keratinocytes, selecting for expression of CD49f, and culturing the CD49f + cells to form the keratinocyte sheet.
  • the functional COL7A1 protein is a full-length wild-type human COL7A1 protein.
  • the functional COL7A1 protein comprises, alternatively consists essentially of, or yet further consists of a genetic modification from a full-length wild-type human COL7A1 protein.
  • the functional COL7A1 protein comprises, alternatively consists essentially of, or yet further consists of a genetic modification from a full-length wild-type human COL7A1 protein, wherein the genetic modification is conservative.
  • the genetic modification comprises, alternatively consists essentially of, or yet further consists of insertion, deletion, and/or mutation.
  • cells are cultured for 1-21 days. In further embodiments, cells are cultured 7, 14, 21 days or longer.
  • cells may be cultured under appropriate conditions for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more days.
  • Cells are re-plated, and media and supplements may be added or changed as needed using techniques known in the art.
  • the genetically altered cells may be cultured under conditions and for sufficient time periods such that at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the cells express the C7 transgene.
  • the cell compositions of the present disclosure comprise, alternatively consist essentially of, or yet further consist of an integration-free genetically altered autologous keratinocyte population, expressing a native human C7 protein in an amount effective for the treatment of EB.
  • Target cell populations are grown in sheets for engraftment onto a subject, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants e.g., chelating agents such as EDTA or glutathione
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • the cells may be administered to the subject by methods well known to those of skill in the art, typically in the form of a skin graft. A medical practitioner will be able to determine a suitable administration route for a particular subject based, in part, on the type and location of the disease.
  • the transfected cells may be administered locally to a wound site.
  • Pharmaceutical preparations of engineered cells for administration to a subject are contemplated by the present invention. One of ordinary skill in the art would be familiar with techniques for administering cells to a subject. Furthermore, one of ordinary skill in the art would be familiar with techniques and pharmaceutical reagents necessary for preparation of these cell sheets prior to administration to a subject.
  • compositions of the present invention comprise an effective amount of a solution of the transfected cells in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutical preparation or “pharmaceutical composition” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the cells, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologics.
  • FDA Center for Biologics A person of ordinary skill in the art would be familiar with techniques for generating sterile solutions for application by any other route. Determination of the size of the cell graft and the number of cells on the graft will be made by one of skill in the art. In certain aspects, multiple doses may be administered over a period of days, weeks, months, or years. A subject may receive, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pieces of graft in the same area or a different area. In one embodiment, the subject may be re-grafted in the same area or a different area.
  • the subject in another embodiment, the subject’s biological sample (e.g., keratinocytes or corneal cells) is stored in proper conditions. Once the biological sample is stored, no punch biopsy is necessary if the subject requires a new graft. The stored biological samples can provide sufficient or supplemental information to determine the graft needed by the subject. [0085] When “an effective amount” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, and condition of the patient (subject).
  • a cell composition comprising the cells described herein may be administered in the amount of 1-100, 1-10 3 , 1-10 4 , 1-10 5 , 1-10 6 , 1-10 7 , or more than 10 7 cells, including all integer values within those ranges. Cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • keratinocytes that are genetically engineered using the methods described herein, or other methods known in the art, are administered to a patient in conjunction with (e.g., before, simultaneously, or following) any number of relevant treatment modalities or delivery devices.
  • Examples [0087] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.
  • Example 1 [0090] RDEB patient keratinocytes contain mutations in the COL7A1 locus, a gene encoding for type VII collagen, the main component of the anchoring fibrils which tether the epidermis to the dermal tissue underneath. From birth, RDEB patients suffer profound skin fragility and an association with delayed wound healing and persistent erosions.
  • the therapeutic strategy is that COL7A1-corrected autologous keratinocyte stem cell sheets, when grafted onto wounds, adhere tightly and provide long-term wound closure.
  • iPSCs can be grown in large quantities and be induced to differentiate into iKCs. Clinical scaling and manufacturing of patient iKC sheets can be achieved by differentiating patient-derived, COL7A1-corrected iPSCs.
  • iPSC-derived RDEB Cell Therapy we developed an alternative strategy to amplify autologous, COL7A1-corrected keratinocyte stem cells called iPSC-derived RDEB Cell Therapy. Starting with patient fibroblasts, DEBCT manufacturing passes through 4 Process Intermediates to generate a final DEBCT graft.
  • the process comprises 7 steps (M1-7), 4 process intermediates (PI#1-4), and the final product.
  • the process is continuous and does not involve any freezing or banking steps from skin biopsy to the skin grafts. A frozen reserve of PI#1,2, and 4 is maintained, in case a later step fails, so we can go back to those intermediates.
  • Step M1 skin biopsy and primary cell culture
  • M7 DEBCT iKC sheet generation and transport to patient
  • Step M2 Reprogram and correct donor cells
  • Step M3 safely expanding corrected iPS intermediate
  • step M4 Disifferentiate Intermediate #3 into iKCs
  • M5 Selecting iKCs by CliniMacs
  • step M6 expansion of iKCs leads to DEBCT production.
  • Step M1 Derivation of RDEB fibroblasts from skin biopsy (PI#1).
  • a skin biopsy is minced using scalpels and chunks of tissue are placed in a tissue culture dish. After 5-9 days fibroblasts grow out of the tissue chunks and populate the dish.
  • the media e.g. DMEM, may be supplemented with 10% FBS.
  • the FBS may be replaced with human serum, HSA, or human platelet lysates. After 8-12 days, the cells are dissociated and subjected to Step M2. In parallel, a small aliquot of fibroblasts is subjected to analysis of release criteria.
  • Step M2 Integration-free, xeno-free, feeder-free single clonal step production of the autologous, CRISPR-corrected iPSC intermediates (PI#2 and #3) from RDEB fibroblasts (PI#1).
  • Manufacturing of COL7A1-corrected autologous iPSCs is optimized through the development of an integration-free, feeder-free, xeno-free, single clonal step method using preformed ribonuclear protein (RNPs) complexes with high-fidelity CAS9 and guide RNAs and mRNAs encoding the Yamanaka reprogramming factors.
  • RNPs ribonuclear protein
  • HR homologous recombination
  • the advantages of this approach include the lack of integrating viruses or clonal selection, and the ability to perform both reprogramming and mutational correction in one step.
  • iPS cell colonies form within 10 days. About ⁇ 100 colonies are picked into 48 wells and upon confluency all re-grown colonies are characterized by droplet digital PCR to quantify COL7A1 correction of the patient mutation via a reference probe that displays a biallelic locus (e.g. RPP30).
  • ddPCR i.e. range of corrected allele frequency 0.4-0.6 in comparison with biallelic reference probe
  • Expanded ddPCR+ colonies are subject to in-depth characterization of the COL7A1 locus by PCR amplification, Sanger sequencing of the PCR product, Topo- cloning and Sanger sequencing of 100 individual PCR amplified loci.
  • Those iPSC lines whose genome yields the expected size PCR product of the amplified edited COL7A1 locus, and whose Sanger sequencing of this edited locus and the unedited locus (i.e. the other COL7A1 allele) shows the expected sequence in 95 out of 100 isolated alleles (i.e. Topo clones)are labeled Process Intermediate #2.
  • a fraction is stored as a reserve and the rest is expanded as outlined below.
  • Step M3 Stable iPS cell expansion to PI#3 in StemFit media.
  • a major problem in the iPSC field is karyotypic (KT) instability during expansion.
  • the standard commercial media available mTESR, E8 etc.
  • mTESR, E8 etc. do not support KT stability in iPSCs over long culture periods. This is likely due to the limited iPSC survival upon splitting, revealing a need for more optimal growth media.
  • a GMP-grade iPSC media that allows iPSC expansion without accumulation of KT abnormalities called “StemFit” in combination with coating the dishes with Laminin 5,1,1 or a fragment of Laminin5,1,1 called E8 fragment performed the best in cell survival and growth and karyotype stability for at least 10 passages.
  • Step M4 Defined, feeder-free, and xeno-free iKC differentiation to PI#4.
  • PI#4 retinoic acid
  • BMP bone morphogenetic protein
  • PI#4 Three major improvements made in manufacturing of iKCs (PI#4) include: Use of embryoid bodies (EB), identification of a E6 defined media that reduces line-to-line variability, and identification of the correlation of ITGA6 surface expression with epidermal stem cells and graftability allowing CliniMACS purification of iKCs.
  • EB embryoid bodies
  • CE Coupling Efficiency
  • Step M5 Purification of graftable iKCs (PI#4) based on CD49f (ITGA6) HI sorting.
  • ITGA6 is one of the earliest surface markers to arise in definitive mature keratinocytes and that ITGA6 bright population correlates with a K14+, K18 - expression basal keratinocyte phenotype. More importantly ITGA6 bright cells displayed enhanced graftability, resulting in an organotypic epidermis that displays proper polarity and differentiation ( Figure 4B-D). By contrast, the unsorted cells contained contaminating immature and undifferentiated cells that were K18 expressing, and generated organotypic cultures with misadherent and mal-oriented keratinocytes. FACS sorting of ITGA6 bright cells and subsequent expansion using CD49f antibodies generate mature iKCs that perform well in organotypic cultures.
  • Step M6 Expansion of selected iKCs after selection in Defined Keratinocyte Media. After selecting early iKCs, we expand iKCs 2-4 passages in keratinocyte defined media DKSFM. We quantify the expansion capabilities of this step by the metric Expansion Coefficient (EC). This media is serum free and low calcium promoting iKC growth and selecting against the growth of other cell types (for example undifferentiated TRA1-60+/SSEA3+ IPSCs).
  • EC Expansion Coefficient
  • Step M7 Forming the graftable epithelial sheet, the DEBCT final product.
  • CD104 the coreceptor for CD49f
  • CD90 expected fibroblast feeders
  • iPSCs undifferentiated iPSCs
  • DEBCT defined by cell composition
  • the final step is the same as the LEAES graft formation and delivery manufacturing step.
  • approximately 5 million iKCs are placed in culture, grown to confluence and then released onto Vaseline gauze and delivered to the operating room (see Figure 1A-C). Wound site selection, graft bed preparation, and grafting of DEBCT onto the patient will follow LEAES IND protocol.
  • Example 2 DEBCT Safety Studies [00105] DEBCT manufacturing requires multiple novel genetic manipulations over a three-month period. We have carried out several assays to demonstrate the genomic integrity and safety of DEBCT. Our major concerns are accumulation of deleterious genetic variations, the presence of undifferentiated pluripotent cells, and the presence of transformed iKCs.
  • Sanger Sequencing provides sensitive detection of COL7A1 variants. To obtain high- quality information about the genomic region in which we are editing, we endeavored to deep sequence the COL7A1 gene around the mutation region. We accomplished this by ddPCR to look at individual cells for mutation correction, by performing qPCR amplification and then sequencing of the region surrounding the mutation, and by subcloning and sequencing 100 clones and determining the frequency of variants. [00107] No Detectable Mutation Selection in the corrected iPS cell intermediate. An important question was whether our long-term cultures selected for particular mutations.
  • the Stanford Oncopanel provides a reliable and meaningful survey of deleterious variants in the genome.
  • NO mutations above the limit of detectability (5%) in any of the intermediates.
  • DPH3 diphthamide synthesis 3
  • the allele frequency suggested that every cell was heterozygous for this mutation. Mutations in the promoter of DPH3 have been associated with the basal cell carcinoma form of keratinocyte tumors.
  • GFP-tagged H9 cells do not incorporate into the DEBCT epithelial sheets in organotypic cultures even if they were present in as high as 50% of input cells.
  • the homotypic desmosome adhesion molecules present on mature keratinocytes prevent H9 incorporation (detection sensitivity 1:100).
  • a two-week expansion in keratinocyte media (step M6) and then DEBCT graft assembly (step M7) reduces the number of iPS cells several logs resulting in too few iPS cells in a clinical dose (30 million iKCs) to cause tumor formation on a surface graft.
  • step M6 A two-week expansion in keratinocyte media (step M6) and then DEBCT graft assembly (step M7) reduces the number of iPS cells several logs resulting in too few iPS cells in a clinical dose (30 million iKCs) to cause tumor formation on a surface graft.
  • Secondary endpoints include comparison of wound healing ⁇ 50% from baseline at 3 months. Wound healing will be assessed and subsequently photographed and quantified using the Canfield system at 0, 3, 6 and 12 months. Exploratory endpoints include Investigator’s Global Assessment (IGA) score (>50%) of each graft/control site at 3 months, patient global impression scale of change at 3 months, and median of PROs assessing the subject’s impression of itch and pain of each graft/control sites at 3 months. Assessment of biological function will include the expression of C7 and the presence of anchoring fibrils and longitudinal changes in PRO itch and pain scores.
  • IGA Global Assessment
  • NC1 domain is generally accepted to be the most antigenic region on the C7 molecule. Therefore, an NC1[+] subject is less likely to develop immune reactions to sites of grafted autologous keratinocytes that express C7.
  • RDEB subjects will be required to express the NC1 amino-terminal fragment of C7 (NC1[+], approximately 75-90% of patients), to be genotyped with confirmed recessive COL7A1 mutations, and to have no evidence of an immune response to C7 by indirect immunofluorescence (IIF).
  • IIF indirect immunofluorescence
  • RDEB subjects will initially be required to be NC1[+], to have absent type VII collagen antibodies, to be genotyped with confirmed recessive COL7A1 mutations, and have no evidence of an immune response to type VII collagen.
  • Subjects could develop physical difficulties which could destroy individual grafts. These events include wound infections or physical trauma which removes the graft before it has attached. We have developed protocols for specific adverse events: immunologic rejection, systemic infection, and advancing epithelial surfaces.
  • Biopsies will be obtained from non- blistered skin for fibroblast culture, in order to manufacture the graft. The manufacturing aim is to produce and deliver up to six of the keratinocyte sheets for grafting (DEBCT).
  • Approximately one to six of the 40-50 cm 2 epithelial sheets will be used in a single grafting session.
  • the maximum total grafting surface area for all the graft sites will be 300 cm 2 .
  • At the screening visit we will select multiple potential wounds for grafting. We will follow these wounds clinically until the day of grafting/enrollment (Day 0). The decision on which sites to be grafted will not be finalized until Day 0. The determination that target wounds meet all graft criteria will be made at that time.
  • the grafted wound areas will be selected by several criteria. The wounds should appear clean with adequate granulation tissue, adequate vascularization, and not appear infected. Bacterial cultures can be obtained from several wounds for culture and antibiotic sensitivities.
  • Wound cultures may be repeated as needed based upon standard of care and medical judgment of the investigators and EB physician.
  • the surface area should have a smooth texture that can accept a graft.
  • the duration that the subject thinks that they have each wound will be recorded.
  • the wounds will also need to meet mechanical requirements that decrease trauma to the grafted areas.
  • Appropriate sites will be on the anterior and/or lateral trunk and/or upper and/or lower extremities in areas protected from frequent trauma or injury. Excluded areas will usually include the face, areas close to mucous membranes (genito- urinary, oral or anal mucosa), areas over joints and back. The distance from objective body landmarks will be identified and measured. [00120] Grafting will be carried out under general anesthesia.
  • Grafts will be labeled with the subject’s name, medical record number (MRN), date of birth (DOB), and study number to confirm that the correct subject is receiving the correct grafts.
  • the grafting process will be: 1. All wounds will be gently cleansed with normal saline or povidone-iodine solution; 2. Overhanging epidermis, hyperkeratotic skin, or fibrinous material will be gently debrided with scalpel, scissors, or the timed surgery electrosurgical technique (or equivalent cauterization technique), or a combination of these at the grafting surgeon’s discretion, in consultation with the EB physician; 3.
  • iKC grafts will be applied to the wound beds and affixed with staples, suture, non-adhesive dressing (Mepitel, Adaptic ® , Restore ® , or other equivalent), and/or overlying dressing.
  • a layer of topical antibiotics will be applied, with specific antibiotic determined by the grafting surgeon and EB physician; 4.
  • subjects will have a small ( ⁇ 1 mm or less) tattoo dot placed at the corners of the grafts and edges of control areas. If the patient does not have useable body landmarks close to the wound site, the PI will choose a location for the surgeon to tattoo a small dot as a landmark.
  • Example 3 iKC differentiation protocol Keratinocyte Differentiation using Embryoid Bodies Protocol Materials AGGREWELL TM 400 (24-well) AGGREWELL TM Medium AGGREWELL TM Rinsing Solution Collagen I peptide 6-well plate Reversible Cell Strainer, 37 ⁇ m Cell strainer, 40 ⁇ m Essential 6 (Gibco) RevitaCell (ROCK Inhibitor,Gibco) 10 cm 2 tissue culture dishes 18 cir-1 coverslips (ThermoFisher) 12-well culture plates Vitronectin (Gibco) Retinoic Acid BMP-4 Petri Dishes Defined Keratinocyte Serum-Free Medium (Gibco) Gentle Cell Dissociation Reagent (StemCell) DMEM/F-12 (Gibco) Preparation.
  • Vitronectin VTN-N, 100X Coated Dishes and 12-well plates with 18 cir-1 coverslips. Coat dishes and 12-well plates with coverslips at least 1 hour before seeding embryoid bodies. Coated dishes and plates can be stored in the cold room wrapped in parafilm for up to 1 week.
  • AggreWell Medium + Revitacell or ROCK inhibitor-1-10 ⁇ M + 1 ⁇ M RA (AG+RV+RA) (100X, RV, ROCK Inhibitor). 24-well requires 2 mL per well, 6-well 5 mL per well.
  • RNA-programmed genome editing in human cells Elife 2, e00471 (2013).
  • Vakulskas, C.A., et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med 24, 1216-1224 (2016).
  • Nakagawa, M., et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4, 3594 (2014).
  • Pattison, J.M., et al. Retinoic acid and BMP4 cooperate with p63 to alter chromatin dynamics during surface epithelial commitment.

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Abstract

L'invention concerne des compositions et des procédés de production de cellules utiles dans des thérapies régénératives.
PCT/US2020/066388 2019-12-23 2020-12-21 Cellules génétiquement corrigées à usage thérapeutique Ceased WO2021133724A1 (fr)

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CN116189765B (zh) * 2023-02-23 2023-08-15 上海捷易生物科技有限公司 一种iPS细胞遗传学风险评估系统及应用

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